Cathepsin B, a lysosomal cysteine endopeptidase, has been implicated in tumor progression, invasion and metastasis. Increased expression (mRNA and protein), membrane/endosomal association and release (latent and mature forms) of cathepsin B are observed in transformed fibroblasts and in malignant murine and human tumors, including human breast, colon, gastric and prostate tumors. In prostate specimens, increases in immunostaining and in situ hybridization for cathepsin B in epithelial cells parallel malignant progression and are observed at the leading, invasive edges of prostate carcinomas. The increase in mRNA and the altered trafficking of cathepsin B in malignant cells probably reflect modifications in more than one step in the normal pathway that leads to the delivery of cathepsin B to lysosomes (i.e., alterations in transcription/translation, co- or post-translational processing, and/or in intracellular sorting and targeting). Multiple mechanisms appear to be responsible for the secretion of cathepsin B since both latent precursor forms and mature forms are released. Although membrane-associated and secreted cathepsin B have been hypothesized to play a role in tumor invasion, there is not yet direct evidence supporting this hypothesis. The studies herein are designed to determine the possible structural bases for the association of cathepsin B with membrane/endosomal fractions and for its secretion and to provide direct evidence for the involvement of cathepsin B in tumor cell invasion. In the first aim of this proposal, we will continue the biochemical characterization of tumor cathepsin B, with an emphasis on those characteristics that could be responsible for the membrane association and secretion of cathepsin B in malignant tumors. The second aim is to examine cathepsin B cDNA clones and genomic sequences from human tumor cell lines for sequence modification. Tumor cathepsin B will be (over)expressed in immortal, non-tumorigenic MCF-10A human breast epithelial cells (Aim 3) and in the poorly invasive ras-transfected MCF-10AneoT cells (Aim 4). These studies will determine whether (over)expression of tumor cathepsin B affects the trafficking and secretion of cathepsin B and/or results in acquisition or enhancement of phenotypic properties associated with malignancy. Properties evaluated will include morphology, growth in low serum and soft agar, invasion, tumorigenicity and metastatic capability. Our working hypothesis is that increased expression of cathepsin B is one factor leading to increases in constitutive and induced secretion of cathepsin B in malignant cells. Upon secretion from malignant cells, cathepsin B can facilitate the invasion of those cells either directly by degrading basement membrane proteins or indirectly via activating other proteolytic enzymes.